As part of efforts to better define the biochemical defects underlying lysosomal storage disorders, we have undertaken studies on the primary structure of several lysosomal enzymes. Initial studies focused on glucocerebrosidase, the enzyme activity deficient in Gaucher's disease, as a prototype for these investigations. In order to complete the amino acid sequence of human placental glucocerebrosidase, peptides were generated by both chemical (cyanogen bromide) and enzymatic (trypsin and V8 protease) cleavage. The peptides were separated by analytical high performance liquid chromatography using reverse-phase columns. The separation of the tryptic digest on the reverse-phase system provided a peptide map which will be useful for studying mutant glucocerebrosidases. The complete amino acid sequence of glucocerebrosidase was determined from the sequence of peptides obtained from the digests. Hence, it was possible to design and prepare oligonucleotide probes from regions of the amino acid sequence which were used to align and confirm the cDNA sequence of the gene cloned for glucocerebrosidase. The secondary structure predicted from the amino acid sequence shows large areas of alpha-helix separated by beta-sheets and turns. Plots of hydropathy reveal alternating stretches of hydrophobic and hydrophilic amino acids throughout the primary structure. Of particular note is the correlation of several stretches of hydrophobicity in regions of Alpha-helical structure. The studies on the primary structure of glucocerebrosidase have revealed the presence of a single free sulfydryl and three disulfide bonds. With the identification of the disulfide bridges, we will construct a model of glucocerebrosidase both in terms of its enzyme activity and its membrane localization, utilizing the secondary structure predictions and hydropathy plots.